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目的 利用已构建的CCND1表达沉默及过表达的人源性胶质母细胞瘤细胞株SHG-44细胞,筛选能够与CCND1协同抑制胶质母细胞瘤细胞增殖的化疗药物。方法 蛋白质印迹法检测CCND1表达沉默及过表达的人源性胶质母细胞瘤细胞株SHG-44中多药耐药基因MDR1表达产物P-糖蛋白(P-gp)及凋亡因子bcl-2、Casepase-3的表达。使用卡莫司汀(BCNU)、洛莫司汀(CCNU)、替莫唑胺(TMZ)3种化疗药物分别处理CCND1过表达或表达沉默的SHG-44细胞,筛选能够与CCND1表达沉默协同抑制肿瘤细胞生长的化疗药物,并在人源性胶质母细胞瘤细胞株系U251中进一步验证。结果 蛋白质印迹结果示CCND1表达沉默能够下调P-gp、Bcl-2表达,上调Casepase-3表达(P<0.05,P<0.01)。BCNU(0.05μg/m L、0.25μg/m L)、CCNU(20μg/m L、80μg/m L)处理CCND1过表达或表达沉默的SHG-44细胞的第2、3、4、5天时,细胞生长曲线的差异均无统计学意义,而在药物处理后细胞培养的第4、5天,CCND1表达沉默SHG-44细胞的生长受到TMZ(9.1μg/m L)的抑制作用较亲代SHG-44细胞强(P<0.05)。U251细胞实验证实CCND1表达沉默促进TMZ的化疗敏感性。结论 CCND1表达沉默联合TMZ较两者分别单独作用能更有效地抑制人源性胶质母细胞瘤细胞株SHG-44的增殖,提示CCND1可能参与TMZ化疗耐药机制。
OBJECTIVE: To screen SHG-44 cells with silenced and over-expressed human glioblastoma cell line CCND1 and screen the chemotherapeutic drugs that can synergistically inhibit the proliferation of glioblastoma cells with CCND1. Methods Western blotting was used to detect the expression of P-glycoprotein (P-gp) and bcl-2 in multidrug resistance gene MDR1 in human glioblastoma cell line SHG-44 with silence of CCND1 expression and overexpression , Casepase-3 expression. The CCND1-overexpressing or silenced SHG-44 cells were treated with 3 chemotherapeutic agents including BCNU, CCNU, and TMZ, respectively, and screened for the synergistic inhibition of tumor cell growth with CCND1 silencing Chemotherapy and further validation in human glioblastoma cell line U251. Results Western blot showed that CCND1 silencing could down-regulate the expression of P-gp and Bcl-2 and up-regulate the expression of Casepase-3 (P <0.05, P <0.01). On the 2nd, 3rd, 4th and 5th days of CCNU overexpression or silenced SHG-44 cells treated with BCNU (0.05μg / mL, 0.25μg / mL), CCNU (20μg / mL, 80μg / mL) However, the growth inhibition of CCND1 expression in SHG-44 cells was inhibited by TMZ (9.1μg / mL) on the 4th and 4th day after drug treatment compared with that of the parental SHG- 44 cells (P <0.05). U251 cell experiments confirmed that CCND1 silencing promotes TMZ chemosensitivity. Conclusion CCND1 silencing combined with TMZ alone can inhibit the proliferation of human glioblastoma cell line SHG-44 more effectively, suggesting that CCND1 may be involved in the chemoresistance mechanism of TMZ chemotherapy.